DNA sequence encoding bovine and human adrenocorticotropic hormone receptors

ABSTRACT

The present invention relates to a mammalian adrenocorticotropic hormone receptor. The invention is directed toward the isolation, characterization and pharmacological use of mammalian adrenocorticotropic hormone receptor, the gene corresponding to this receptor, a recombinant eukaryotic expression construct capable of expressing a mammalian adrenocorticotropic hormone receptor in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize mammalian adrenocorticotropic hormone receptor. The invention also provides methods for screening ACTHR agonists and antagonists in vitro using preparations of receptor from such cultures of eukaryotic cells transformed with a recombinant eukaryotic expression construct comprising the ACTHR receptor gene. The invention specifically provides human and bovine ACTHR genes.

BACKGROUND OF THE INVENTION

This invention was made with government support under 1R01DK41921-03,1R01DK43859-01, and 1P01DK44239-10A1 by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to adrenocorticotropic hormone receptors frommammalian species and the genes corresponding to such receptors.Specifically, the invention relates to the isolation, cloning andsequencing of a human adrenocorticotropic hormone receptor gene. Theinvention also relates to the isolation, cloning and sequencing of abovine adrenocorticotropic hormone receptor gene. The invention relatesto the construction of eukaryotic recombinant expression constructscapable of expressing these adrenocorticotropic hormone receptors incultures of transformed eukaryotic cells, and the production of theadrenocorticotropic hormone receptor in such cultures. The inventionrelates to the use of such cultures of transformed eukaryotic cells toproduce homogeneous compositions of such adrenocorticotropic hormonereceptors. The invention also provides culture of such cells producingadrenocorticotropic hormone receptor for the characterization of noveland useful drugs.

BACKGROUND OF THE INVENTION

The proopiomelanocortin (POMC) gene product is processed to produce alarge number of biologically active peptides. Two of these peptides,alpha-adrenocorticotropic hormone (αMSH), and adrenocorticotropichormone (ACTH) have well understood roles in control of melanocyte andadrenocortical function, respectively. Both of these hormones, however,are found in a variety of forms with unknown functions. The melanocortinpeptides also have a diverse array of biological activities in othertissues, including the brain and immune system, and bind to specificreceptors there with a distinct pharmacology [see, Hanneman et al., inPeptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.:Chichester, UK) pp. 53-82; DeWied & Jolles, 1982, Physiol. Rev. 62:976-1059 for reviews].

A complete understanding of these peptides and their diverse biologicalactivities requires the isolation and characterization of theircorresponding receptors. Some biochemical studies have been reported onthe prior art.

Oelofsen & Ramachandran, 1983, Arch. Biochem. Biophys. 225: 414-421disclose receptor binding studies on ACTH receptors on rat adipocytes.

Mertz & Catt, 1991, Proc. Natl. Acad. Sci. USA 88:8525-8529 disclosefunctional expression of ACTH receptors in Xenopus laevis oocytesfollowing injection of total cellular RNA from adrenal tissue.

Moore et al., 1991, Endocrinology 34: 107-114 relates to Allgrovesyndrome, an autosomal recessive syndrome characterized by ACTHinsensitivity.

The present invention comprises a human adrenocorticotropic hormonereceptor gene, the nucleotide sequence of this gene and the deducedamino acid sequence of its cognate protein, a homogeneous composition ofthe adrenocorticotropic hormone receptor, nucleic acid hybridizationprobes and a method for determining the tissue distribution ofexpression of the gene, a recombinant expression construct capable ofexpressing the gene in cultures of transformed eukaryotic cells, andsuch cultures of transformed eukaryotic cells useful in thecharacterization of novel and useful drugs. The present invention alsocomprises the bovine adrenocorticotropic hormone receptor gene.

FIG. 1A-1C illustrates the nucleotide sequence of the human (SEQ IDNO:3) adrenocorticotropic hormone receptor.

FIG. 2 illustrates the nucleotide sequence of the bovine (SEQ ID NO:5)adrenocorticotropic hormone receptor.

FIG. 3 presents an amino acid sequence comparison between the humanadrenocorticotropic hormone receptor protein and the mouse and humanmelanocyte stimulating hormone receptor proteins.

FIG. 4 illustrates the tissue distribution of human adrenocorticotropichormone receptor gene expression by Northern blot hybridization.

FIG. 5 illustrates localization of the putative ACTH receptor mRNA tothe adrenal cortex by in situ hybridization (brightfield illumination).

FIG. 6 illustrates localization of the putative ACTH receptor mRNA tothe adrenal cortex by in situ hybridization (darkfield illumination).

SUMMARY OF THE INVENTION

The present invention relates to the cloning, expression and functionalcharacterization of mammalian adrenocorticotropic hormone receptor(ACTH^(R)) genes. The invention comprises the nucleotide sequence ofthese genes encoding the mammalian ACTH^(R) s and the deduced amino acidsequences of the cognate proteins, as well as tissue distributionpatterns of expression of these genes.

In particular, the present invention is directed toward the isolation,characterization and pharmacological use of the human ACTH^(R), the genecorresponding to this receptor, a nucleic acid hybridization probecomprising DNA sequences of the human ACTH^(R), a recombinant eukaryoticexpression construct capable of expressing the human ACTH^(R) incultures of transformed eukaryotic cells and such cultures oftransformed eukaryotic cells that synthesize the human ACTH^(R), ahomogeneous composition of the human ACTH^(R), and antibodies againstand epitopes of the human ACTH^(R).

The present invention is also directed toward the isolation,characterization and pharmacological use of the bovine ACH^(R), the genecorresponding to this receptor, a nucleic acid hybridization probecomprising DNA sequences of the bovine ACTH^(R), a recombinanteukaryotic expression construct capable of expressing the bovineACTH^(R) in cultures of transformed eukaryotic cells and such culturesof transformed eukaryotic cells that synthesize the bovine ACTH^(R), ahomogeneous composition of the bovine ACTH^(R), and antibodies againstand epitopes of the bovine ACTH^(R).

It is an object of the invention to provide a nucleotide sequenceencoding a mammalian ACTH^(R). In a preferred embodiment of theinvention, the nucleotide sequence encodes the human ACTH^(R). Inanother preferred embodiment, the nucleotide sequence encodes the bovineACTH^(R).

The present invention includes a nucleotide sequence encoding a humanACTH^(R) receptor derived from a DNA molecule isolated from a humangenomic library (SEQ ID NO:5). In this embodiment of the invention, thenucleotide sequence includes 2028 nucleotides of the human ACTH^(R) genecomprising 893 nucleotides of coding sequence, 696 nucleotides of 5'untranslated sequence and 439 nucleotides of 3' untranslated sequence.

The present invention also includes a nucleotide sequence encoding abovine ACTH^(R) R derived from a cDNA molecule isolated from a cDNAlibrary constructed with bovine RNA (SEQ ID NO:3). In this embodiment ofthe invention, the nucleotide sequence includes 1106 nucleotides of thebovine ACTH^(R) gene comprising 893 nucleotides of coding sequence, 133nucleotides of 5' untranslated sequence and 82 nucleotides of 3'untranslated sequence.

The invention includes nucleotide sequences of mammalian ACTH^(R) s,most preferably bovine and human ACTH^(R) s (SEQ ID NOs:3 and 5), andincludes allelic variations of these nucleotide sequences and thecorresponding ACTH^(R) molecule, either naturally occurring or theproduct of in vitro chemical or genetic modification, each such varianthaving essentially the same nucleotide sequence as the nucleotidesequence of the corresponding ACTH^(R) disclosed herein, wherein theresulting ACTH^(R) molecule has substantially the same biologicalproperties as the ACTH^(R) molecule corresponding to the nucleotidesequence described herein. The term "substantially homologous to" asused in this invention encompasses such allelic variability as describedin this paragraph.

The invention also includes a predicted amino acid sequence for thebovine (SEQ ID NO:4) and human (SEQ ID NO:6) ACTH^(R) deduced from thenucleotide sequence comprising the complete coding sequence of thebovine (SEQ ID NO:3) and human (SEQ ID NO:5) ACTH^(R) R gene asdescribed herein.

In another aspect, the invention comprises a homogeneous composition ofa 34 kilodalton bovine ACTH^(R) or derivative thereof, wherein the aminoacid sequence of the ACTH^(R) or derivative thereof comprises a sequenceshown in FIG. 3 (SEQ ID NO:4).

In another aspect, the invention comprises a homogeneous composition ofa 34 kilodalton human ACTH^(R) or derivative thereof, wherein the aminoacid sequence of the ACTH^(R) or derivative thereof comprises a sequenceshown in FIG. 3 (SEQ ID NO:6).

This invention provides both nucleotide and amino acid probes derivedfrom these sequences. The invention includes probes isolated from eithercDNA or genomic DNA clones, as well as probes made synthetically withthe sequence information derived therefrom. The invention specificallyincludes but is not limited to oligonucleotide, nick-translated, randomprimed, or in vitro amplified probes made using cDNA or genomic cloneembodying the invention, and oligonucleotide and other synthetic probessynthesized chemically using the nucleotide sequence information of cDNAor genomic clone embodiments of the invention.

It is a further object of this invention to provide sequences ofmammalian ACTH^(R), preferably the bovine or human ACTH^(R), for use asnucleic acid hybridization probes to determine the pattern, amount andextent of expression of this receptor in various tissues of mammals,including humans. It is also an object of the present invention toprovide nucleic acid hybridization probes derived from the sequences ofthe bovine or human ACTH^(R) to be used for the detection and diagnosisof genetic diseases. It is an object of this invention to provide nucleiacid hybridization probes derived from the DNA sequences of the bovineor human ACTH^(R) to be used for the detection of novel related receptorgenes.

The present invention also includes synthetic peptides made using thenucleotide sequence information comprising cDNA or genomic cloneembodiments of the invention. The invention includes either naturallyoccurring or synthetic peptides which may be used as antigens for theproduction of ACTH^(R) -specific antibodies, or used for competitors ofthe ACTH^(R) molecule for drug binding, or to be used for the productionof inhibitors of the binding of agonists or antagonists or analoguesthereof to ACTH^(R) molecule.

The present invention also provides antibodies against and epitopes ofmammalian ACTH^(R) s, preferably bovine or human ACTH^(R) proteins. Itis an object of the present invention to provide antibodies that isimmunologically reactive to a mammalian ACTH^(R) protein. It is aparticular object of the invention to provide a monoclonal antibodies tomammalian ACTH^(R) protein, most preferably bovine or human ACTH^(R)protein.

It is also an object of the present invention to provide a hybridomacell line that produces such an antibody. It is a particular object ofthe invention to provide a hybridoma cell line that is the result offusion between a non-immunoglobulin producing bovine myeloma cell lineand spleen cells derived from a bovine immunized with a human cell linewhich expresses ACTH^(R) antigen. The present invention also provides ahybridoma cell line that produces such an antibody, and that can beinjected into a living bovine to provide an ascites fluid from thebovine that is comprised of such an antibody.

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a monoclonal antibodythat is immunologically reactive to a mammalian ACTH^(R), preferably abovine or human ACTH^(R) R, and in a pharmaceutically acceptablecarrier.

It is a further object of the present invention to provide an epitope ofa mammalian ACTH^(R) protein wherein the epitope is immunologicallyreactive to an antibody specific for the mammalian ACTH^(R). Inpreferred embodiments, the epitope is derived from bovine of humanACTH^(R) protein.

It is another object of the invention to provide a chimeric antibodythat is immunologically reactive to a mammalian ACTH^(R) protein. In apreferred embodiment, the chimeric antibody is a monoclonal antibody. Ina preferred embodiment, the ACTH^(R) is a bovine or human ACTH^(R).

The present invention provides a recombinant expression constructcomprising the nucleotide sequence of a mammalian ACTH^(R), preferablythe bovine or human ACTH^(R) and sequences sufficient to direct thesynthesis of bovine or human ACTH^(R) in cultures of transformedeukaryotic cells. In a preferred embodiment, the recombinant expressionconstruct is comprised of plasmid sequences derived from the plasmidpcDNAI/neo and cDNA or genomic DNA of bovine or human ACTH^(R) gene.This invention includes a recombinant expression construct comprisingessentially the nucleotide sequences of genomic or cDNA clones of bovineor human ACTH^(R) in an embodiment that provides for their expression incultures of transformed eukaryotic cells.

It is also an object of this invention to provide cultures oftransformed eukaryotic cells that have been transformed with such arecombinant expression construct and that synthesize mammalian,preferably bovine or human, ACTH^(R) protein. In a preferred embodiment,the invention provides human 293 cells that synthesize bovine ACTH^(R).In an additional preferred embodiment, the invention provides human 293cells that synthesize human ACTH^(R) protein.

The present invention also includes protein preparations of mammalian,preferably bovine or human ACTH^(R), and preparations of membranescontaining mammalian ACTH^(R), derived from cultures of transformedeukaryotic cells. In a preferred embodiment, cell membranes containingbovine ACTH^(R) protein are isolated from 293 cell cultures transformedwith a recombinant expression construct that directs the synthesis ofbovine ACTH^(R). In another preferred embodiment, cell membranescontaining human ACTH^(R) protein are isolated from 293 cell culturestransformed with a recombinant expression construct that directs thesynthesis of human ACTH^(R).

It also an object of this invention to provide mammalian, preferablybovine or human ACTH^(R) for use in the in vitro screening of noveladenosine agonist and antagonist compounds. In a preferred embodiment,membrane preparations containing the bovine ACTH^(R), derived fromcultures of transformed eukaryotic cells, are used to determine the drugdissociation properties of various novel adenosine agonist andantagonist compounds in vitro. In another preferred embodiment, membranepreparations containing the human ACTH^(R), derived from cultures oftransformed eukaryotic cells, are used to determine the drugdissociation properties of various novel adenosine agonist andantagonist compounds in vitro. These properties are then used tocharacterize such novel compounds by comparison to the bindingproperties of known bovine or human ACTH^(R) agonists and antagonists.

The present invention is also useful for the in vivo detection ofanalogues of agonists or antagonists of ACTH^(R), known or unknown,either naturally occurring or as the embodiments of a drug.

It is an object of the present invention to provide a method for thequantitative detection of agonists or antagonists, or analogues thereof,of ACTH^(R), known or unknown, either naturally occurring or as theembodiments of a drug. It is an additional object of the invention toprovide a method to detect such agonists, antagonists, or analoguesthereof in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, orany other bodily fluid.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "adrenocorticotropic hormone receptor" as used herein refers toproteins substantially homologous to, and having substantially the samebiological activity as, the protein coded for by the nucleotide sequencedepicted in FIGS. 1A-1C (SEQ ID NO:3). This definition is intended toencompass natural allelic variations in the adrenocorticotropic hormonereceptor sequence. Cloned genes of the present invention may code forACTH^(R) s of any species of origin, including, for example, bovine,rat, rabbit, cat, and human, but preferably code for receptors ofmammalian, most preferably bovine and human, origin.

Nucleic acid hybridization probes provided by the invention comprise DNAsequences that are substantially homologous to the DNA sequences inFIGS. 1A-1C (SEQ ID NO:3) and 2 (SEQ ID NO:5). Nucleic acid probes areuseful for detecting ACTH^(R) gene expression in cells and tissues usingtechniques well-known in the art, including but not limited to Northernblot hybridization, in situ hybridization and Southern hybridization toreverse transcriptase - polymerase chain reaction product DNAs. Theprobes provided by the present invention, including oligonucleotidesprobes derived therefrom, are useful are also useful for Southernhybridization of mammalian, preferably human, genomic DNA for screeningfor restriction fragment length polymorphism (RFLP) associated withcertain genetic disorders.

The production of proteins such as the ACTH^(R) from cloned genes bygenetic engineering is well known. See, e.g., U.S. Pat. No. 4,761,371 toBell et al. at Col. 6 line 3 to Col. 9 line 65. (The disclosure of allU.S. patent references cited herein is to be incorporated herein byreference.) The discussion which follows is accordingly intended as anoverview of this field, and is not intended to reflect the full state ofthe art.

DNA which encodes the ACTH^(R) may be obtained, in view of the instantdisclosure, by chemical synthesis, by screening reverse transcripts ofmRNA from appropriate cells or cell line cultures, by screening genomiclibraries from appropriate cells, or by combinations of theseprocedures, as illustrated below. Screening of mRNA or genomic DNA maybe carried out with oligonucleotide probes generated from the ACTH^(R)gene sequence information provided herein. Probes may be labeled with adetectable group such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with known procedures and used inconventional hybridization assays, as described in greater detail in theExamples below. In the alternative, ACTH^(R) gene sequences may beobtained by use of the polymerase chain reaction (PCR) procedure, withthe PCR oligonucleotide primers being produced from the ACTH^(R) R genesequence provided herein. See U.S. Pat. No. 4,683,195 to Mullis et al.and U.S. Pat. No. 4,683,202 to Mullis.

The ACTH^(R) may be synthesized in host cells transformed with arecombinant expression construct comprising a DNA sequence encoding theACTH^(R). Such a recombinant expression construct can also be comprisedof a vector that is a replicable DNA construct. Vectors are used hereineither to amplify DNA encoding the ACTH^(R) and/or to express DNA whichencodes the ACTH^(R). For the purposes of this invention, a recombinantexpression construct is a replicable DNA construct in which a DNAsequence encoding the ACTH^(R) is operably linked to suitable controlsequences capable of effecting the expression of the ACTH^(R) in asuitable host. The need for such control sequences will vary dependingupon the host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation. Amplification vectors donot require expression control domains. All that is needed is theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants.

Vectors useful for practicing the present invention includes plasmids,viruses (including phage), retroviruses, and integratable DNA fragments(i.e., fragments integratable into the host genome by homologousrecombination). The vector replicates and functions independently of thehost genome, or may, in some instances, integrate into the genomeitself. Suitable vectors will contain replicon and control sequenceswhich are derived from species compatible with the intended expressionhost. A preferred vector is the plasmid pcDNAI/neo. Transformed hostcells are cells which have been transformed or transfected withrecombinant expression constructs made using recombinant DNA techniquesand comprising a mammalian ACTH^(R). Transformed host cells mayordinarily express the mammalian ACTH^(R), but host cells transformedfor purposes of cloning or amplifying nucleic acid hybridization probeDNA need not express the receptor. When expressed, the mammalianACTH^(R) will typically be located in the host cell membrane.

DNA regions are operably linked when they are functionally related toeach other. For example: a promoter is operably linked to a codingsequence if it controls the transcription of the sequence; a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to permit translation. Generally, operably linked means contiguousand, in the case of leaders sequences, contiguous and in the sametranslational reading frame.

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant ACTH^(R) synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. However, mammalian cells are preferred, asillustrated in the Examples. Propagation of such cells in cell culturehas become a routine procedure. See Tissue Culture, Academic Press,Kruse & Patterson, editors (1973). Examples of useful host cell linesare human 293 cells, VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Human 293cells are preferred. Expression vectors for such cells ordinarilyinclude (if necessary) an origin of replication, a promoter locatedupstream from the gene to be expressed, along with a ribosome bindingsite, RNA splice sites (if intron-containing genomic DNA is used), apolyadenylation site, and a transcriptional termination sequence.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral source (e.g., polyoma, adenovirus, VSV, or MPV), or maybe provided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient.

The invention provides homogeneous compositions of mammalian ACTH^(R)protein produced by transformed eukaryotic cells as provided herein.Such homogeneous compositions are intended to be comprised of mammalianACTH^(R) protein that comprises 90% of the protein in such homogenouscomposition.

Mammalian ACTH^(R) protein made form cloned genes in accordance with thepresent invention may be used for screening agonist compounds forACTH^(R) activity, or for determining the amount of a ACTH^(R) agonistor antagonist drug in a solution (e.g., blood plasma or serum). Forexample, host cells may be transformed with a recombinant expressionconstruct of the present invention, ACTH^(R) expressed in that host, thecells lysed, and the membranes from those cells used to screen compoundsfor ACTH^(R) binding activity. Competitive binding assays in which suchprocedures may be carried out are well known in the art. By selection ofhost cells which do not ordinarily express ACTH^(R) s, pure preparationsof membranes containing ACTH^(R) s can be obtained. Further, ACTH^(R)agonists and antagonists can be identified by transforming host cellswith vectors of the present invention. Membranes obtained from suchcells can be used in binding studies wherein the drug dissociationactivity is monitored.

The recombinant expression constructs of the present invention areuseful in molecular biology to transform cells which do not ordinarilyexpress the ACTH^(R) to thereafter express this receptor. Such cells areuseful as intermediates for making cell membrane preparations useful forreceptor binding assays, which are in turn useful for drug screening.Further, genes and vectors comprising the recombinant expressionconstruct of the present invention are useful in gene therapy. For suchpurposes, retroviral vectors as described in U.S. Pat. No. 4,650,764 toTemin & Watanabe or U.S. Pat. No. 4,861,719 to Miller may be employed.Cloned genes of the present invention, or fragments thereof, may also beused in gene therapy carried out homologous recombination orsite-directed mutagenesis. See generally Thomas & Capecchi, 1987, Cell51: 503-512; Bertling, 1987, Bioscience Reports 7: 107-112; Smithies etal., 1985, Nature 317: 230-234.

Oligonucleotides of the present invention are useful as diagnostic toolsfor probing ACTH-receptor gene expression in tissues. For example,tissues can be probed in situ with oligonucleotides probes carryingdetectable groups by conventional autoradiography techniques, asexplained in greater detail in the Examples below, to investigate nativeexpression of this receptor or pathological conditions relating thereto.Further, chromosomes can be probed to investigate the presence orabsence of the ACTH^(R) gene, and potential pathological conditionsrelated thereto, as also illustrated by the Examples below.

The invention also provides antibodies that are immunologically reactiveto a mammalian ACTH^(R). The antibodies provided by the invention can beraised in animals by inoculation with cells that express a mammalianACTH^(R) R or epitopes of a mammalian ACTH^(R) using methods well knownin the art. Animals that can be used for such inoculations includeindividuals from species comprising cows, sheep, pigs, mice, rats,rabbits, hamsters, goats and primates. Preferred animals for inoculationare rodents (including mice, rats, hamsters) and rabbits. The mostpreferred animal is the mouse.

Cells that can be used for such inoculations, or for any of the othermeans used in the invention, include any cell line which naturallyexpresses a mammalian ACTH^(R), or any cell or cell line that expressesa mammalian ACTH^(R) or any epitope therein as a result of molecular orgenetic engineering, or that has been treated to increase the expressionof a mammalian ACTH^(R) by physical, biochemical or genetic means.Preferred cells are human cells, most preferably human 293 cells thathave been transformed with a recombinant expression construct comprisingDNA sequences encoding a mammalian ACTH^(R) and that express themammalian ACTH^(R) gene product.

The present invention provides monoclonal antibodies that areimmunologically reactive with an epitope that is a mammalian ACTH^(R)present on the surface of mammalian cells, preferably human or bovinecells. These antibodies are made using methods and techniques well knownto those of skill in the art.

Monoclonal antibodies provided by the present invention are produced byhybridoma cell lines, that are also provided by the invention and thatare made by methods well known in the art. Hybridoma cell lines are madeby fusing individual cells of a myeloma cell line with spleen cellsderived from animals immunized with cells expressing a mammalianACTH^(R), including human cells, as described above. The myeloma celllines used in the invention include lines derived from myelomas of mice,rats, hamsters, primates and humans. Preferred myeloma cells lines arefrom bovine, and the most preferred bovine myeloma cell line isP3X63-Ag8.653. The animals from whom spleens are obtained afterimmunization are rats, mice and hamsters, preferably mice, mostpreferably Balb/c mice. Spleen cells and myeloma cells are fused using anumber of methods well known in the art, including but not limited toincubation with inactivated Sendai virus and incubation in the presenceof polyethylene glycol (PEG). The most preferred method for cell fusionis incubation in the presence of a solution of 45% (w/v) PEG-1450.Monoclonal antibodies produced by hybridoma cell lines can be harvestedfrom cell culture supernatant fluids from in vitro cell growth;alternatively, hybridoma cells can be injected subcutaneously and/orinto the peritoneal cavity of an animal, most preferably a bovine, andthe monoclonal antibodies obtained from blood and/or ascites fluid.

Monoclonal antibodies provided by the present invention can also beproduced by recombinant genetic methods well known to those of skill inthe art, and the present invention encompasses antibodies made by suchmethods that are immunologically reactive with an epitope of a mammalianACTH^(R).

The present invention encompasses fragments of the antibody that areimmunologically reactive with an epitope of a mammalian ACTH^(R). Suchfragments can be produced by any number of methods, including but notlimited to proteolytic cleavage, chemical synthesis or preparation ofsuch fragments by means of genetic engineering technology. The presentinvention also encompasses single-chain antibodies that areimmunologically reactive with an epitope of a mammalian ACTH^(R) made bymethods known to those of skill in the art.

The present invention also encompasses an epitope of a mammalianACTH^(R) that is comprised of sequences and/or a conformation ofsequences present in the mammalian ACTH^(R) molecule. This epitope maybe naturally occurring, or may be the result of proteolytic cleavage ofthe mammalian ACTH^(R) molecule and isolation of an epitope-containingpeptide or may be obtained by synthesis of an epitope-containing peptideusing methods well known to those skilled in the art. The presentinvention also encompasses epitope peptides produced as a result ofgenetic engineering technology and synthesized by genetically engineeredprokaryotic or eukaryotic cells.

The invention also includes chimeric antibodies, comprised ofimmunologically reactive light chain and heavy chain peptides to anepitope that is a mammalian ACTH^(R). The chimeric antibodies embodiedin the present invention include those that are derived from naturallyoccurring antibodies as well as chimeric antibodies made by means ofgenetic engineering technology well known to those of skill in the art.

The Examples which follow are illustrative of specific embodiments ofthe invention, and various uses thereof. They are set forth forexplanatory purposes only, and are not to be taken as limiting theinvention.

EXAMPLE 1 Isolation of an ACTH Receptor Probe by Random PCRAmplification of Human Melanoma cDNA Using Degenerate OligonucleotidePrimers

In order to clone novel G-protein coupled receptors, human melanoma cDNAwas used as template for a polymerase chain reaction (PCR)-based randomcloning experiment. PCR was performed using a pair of degenerateoligonucleotide primers corresponding to the putative third and sixthtransmembrane regions of G-protein coupled receptors (Libert et al.,1989, Science 244: 569-72; Zhou et al., 1990, Nature 347: 76-80). ThePCR products obtained in this experiment were characterized bynucleotide sequencing. Two novel sequences representing novelG-protein-coupled receptors were identified.

PCR amplification was performed as follows. Total RNA was isolated froma human melanoma tumor sample by the guanidinium thiocyanate method(Chirgwin et al., 1979, Biochemistry 18: 5294-5299). Double-strandedcDNA was synthesized from total RNA with murine reverse transcriptase(BRL, Gaithersburg, Md.) by oligo-dT priming [Maniatis et al., MolecularCloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), 1990]. The melanoma cDNA mixture was thensubjected to 45 cycles of PCR amplification using 500 picomoles ofdegenerate oligonucleotide primers having the following sequence:##STR1## in 100 μl of a solution containing 50 mM Tris-HCl (pH 8.3), 2.5mM MgCl₂, 0.01% gelatin, 200 μM each dNTP, and 2.5 units of Taqpolymerase (Saiki et al., 1988, Science 239: 487-491). These primerswere commercially synthesized by Research Genetics Inc. (Huntsville,Ala.). Each PCR amplification cycle consisted of incubations at 94° C.for 1 min (denaturation), 45° C. for 2 min (annealing), and 72° C. for 2min (extension).

Amplified products of the PCR reaction were extracted withphenol/chloroform and precipitated with ethanol. After digestion withEcoRI and SalI, the PCR products were separated on a 1.2% agarose gel. Aslice of this gel, corresponding to PCR products of 300 basepairs (bp)in size, was cut out and purified using glass beads and sodium iodide,and then the insert was cloned into a pBKS cloning vector (Stratagene,LaJolla, Calif.).

A total of 172 of such pBKS clones containing inserts were sequencedusing Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) by thedideoxynucleotide chain termination method (Sanger et al., 1977, Proc.Natl. Acad. Sci. USA 74: 5463-5467). Two types of sequences homologousto other G-protein coupled receptors were identified.

EXAMPLE 2 Isolation and Characterization of Human ACTH^(R) GenomicClones

In order to isolate the human gene corresponding to one of the twoG-protein coupled receptor probes of Example 1, a human genomic librarywas screened at high stringency (50% formamide, 1M NaCl, 50 nM Tris-HCl,pH 7.5, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100μg/ml salmon sperm DNA, 10X Denhardt's solution, 42° C.), using thehuman PCR fragments isolated as described in Example 1. Two differenttypes of sequences were isolated, corresponding to the two PCRfragments, and were found to encode highly related G protein coupledreceptors. These genomic clones were sequences as described inExample 1. The nucleotide sequence of this clone is shown in FIGS. 1A-1C(SEQ ID NO:3). Nucleotide sequence analysis and homology comparisonswere done on the OHSU computer system with software provided byIntelligenetics Inc. (Mountain View, Calif).

One of these genomic clones were determined to encode an human MSHreceptor. The human MSH receptor has a predicted amino acid sequencethat is 75% identical and colinear with a mouse αMSH receptor cDNAsequence.

The second human genomic clone isolated encodes a highly relatedG-coupled receptor protein (SEQ ID NO:3). The predicted amino acidsequence (SEQ ID NO:4) of this clone (FIG. 3, represented as humanACTH-R) is 39% identical and also colinear, excluding the thirdintracellular loop and carboxy-terminal tail, with the human MSHreceptor gene product (FIG. 3; represented as human MSH-R). Thepredicted molecular weight of this putative ACTH^(R) is 33.9 kilodaltons(kD). Based on its high degree of homology to the murine (mouse MSH-R;FIG. 3) and human MSH receptors, and the pattern of expression indifferent tissue types, as described in Example 3 below, this gene is abelieved to encode a human ACTH receptor.

A bovine genomic DNA clone was isolated from a bovine genomic library,essentially as described above, and its nucleotide sequence determined(FIG. 2; SEQ ID NO:5).

The predicted amino acid sequences of the mouse αMSH^(R), human MSH^(R),and the putative human ACTH^(R) are aligned in FIG. 3. These sequencesdefine the melanocortin receptors as a novel subfamily of the Gprotein-coupled receptors with a number of unusual features. Themelanocortin receptors rae the smallest G protein-coupled receptorsidentified to date (297-317aa) resulting from a short amino terminalextracellular domain, a short carboxy-terminal intracellular domain, anda very small third intracellular loop. The melanocortin receptors arelack several amino acid residues present in most G protein coupledreceptors (see Probst et al., 1992, DNA & Cell Biol. 11: 1-20),including the proline residues in the 4th and 5th transmembrane domains,likely to introduce a bend in the alpha helical structure of thetransmembrane domains and thought to be involved in the formation of thebinding pocket (see Applebury & Hargrave, 1986, Vision Res. 26:1881-1895), and one or both of the cysteine residues though to form adisulfide bond between the first and second extracellular loops (seeDixon et al., 1987, EMBO J. 6: 3269-3275 and Karnik et al., 1988, Proc.Natl. Acad. Sci. USA 85:8459-8463). Remarkably, the melanocortinreceptors do not appear highly related to the other G protein-coupledreceptors which recognize peptide ligands, such as the receptors forbombesin (see Spindel et al., 1990, Mol. Endocrinol. 4: 1956-1963) orsubstance K (see Masu et al., 1987, Nature 329: 836-838), but rather,are more closely related to the receptor for Δ⁹ -tetradhydrocannabinol(see Matsuda et al., 1990, Nature 346: 561-564). For example, the humanACTH^(R) and rat cannabinoid receptors are about 30% identical inpredicted transmembrane and intracellular loop amino acid sequences. Thecannabinoid receptor also lacks the conserved proline in transmembrane 5and the cysteine in the first extracellular loop necessary for disulfidebond formation. Least parsimony analysis with the receptor sequencesshown in FIG. 3 suggests the cannabinoid and melanocortin receptors maybe evolutionarily related and form a subfamily distinct from the peptidereceptors and the amine receptors. Regardless of whether thesimilarities are the result of evolutionary conservation or convergence,the sequence and putative structural similarities between themelanocortin and cannabinoid receptors may be informative in the searchfor the endogenous cannabinoid-like ligand.

EXAMPLE 3 Tissue Distribution of ACTH Receptor Gene Expression

To further gain insight into this receptor, we have examined the tissuedistribution of its corresponding mRNA from various tissues byperforming Northern hybridization experiments on RNA isolated fromvarious tissues (see Maniatis et al., ibid.). The results of theseexperiments are shown in FIG. 4.

A panel of tissue samples was examined by Northern hybridizationanalysis performed under high stringency conditions. The nitrocellulosefilter was hybridized with a putative human ACTH receptor probe todetermine the distribution of receptor mRNA. In two primary humanmelanocyte cultures examined, the ACTH^(R) is encoded by two mRNAspecies of approximately equal stiochiometry, one at 3.0 kb, and onewhich co-migrates with murine αMSH^(R) mRNA at 3.9 kb.

The putative human ACTH receptor is encoded predominantly by a singlemRNA species of approximately 4.0 kb in the human adrenal gland,although several minor species are present as well. Northern analysis ofa panel of tissues from the rhesus macaque performed under highstringency conditions demonstrated the existence of a cross-reacting 4.0kb species specific to the rhesus adrenal gland (FIG. 4). In situhybridization of a fragment of the putative human ACTH receptor tosections of rhesus adrenal tissue localized the expression of thisreceptor solely to the cortex, with no apparent hybridization to themedulla or capsule, as would be predicted for this receptor (FIG. 5-6).Adrenal tissue from a juvenile rhesus macaque was fixed for 24 hours in10% formalin in phosphate buffered saline, then incubated for 24 hoursin 20% sucrose in PBS. Sections were prepared and hybridized with a 600nucleotide ³⁵ S-labelled RNA antisense probe complementary to codingsequence spanning transmembrane domains 1-6 of the putative human ACTHreceptor. Hybridizations were performed at 65° C. in 2× SSC and washedat 65° C. with 0.1×SSC.

The results of these experiments are shown in FIG. 5-6. FIG. 5illustrates lightfield micrograph of an hematoxylin and eosin stainedsection of rhesus adrenal showing capsule (C), zona glomerulosa (G),zona fasciculata (F), zona reticulate (R), and medulla (M). FIG. 6depicts darkfield micrograph of the same field. Within the cortex,receptor expression was found across the entire zona fasciculata, thesite of glucocorticoid production, and in the cortical half of the zonaglomerulosa, the site of aldosterone synthesis. The zona reticulata waslargely negative, except for a small band of hybridization adjacent tothe medulla, which might result from a cross-reaction between theputative ACTH^(R) probe and a receptor for γ₃ MSH, which is known tobind to this region of the adrenal cortex.

Additionally, we have been unable to detect expression in the brain ofACTH receptor described here, despite extensive documentation of ACTHbinding sites there as well as in other tissues. These finding suggestthe existence of alternate forms of these or related receptors that maybe specifically expressed in brain tissue.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A ) NAME/KEY: miscfeature                                                    (B) LOCATION: 1..33                                                           (D) OTHER INFORMATION: /function="Degenerate                                  oligonucleotide primer (sense)"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGTCGACCTGTGYGYSATYRCTKGACMGSTAC33                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iv) ANTI-SENSE: YES                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..31                                                           (D) OTHER INFORMATION: /function="Degenerate                                  oligonucleotide primer (antisense)"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGAATTCAGWAGGGCA CCAGCAGASRYGAA31                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2012 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                              (B) LOCATION: 694..1587                                                      (D) OTHER INFORMATION: /product="Human                                        adrenocorticotropic hormone receptor"                                         (ix) FEATURE:                                                                 (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..693                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1588..2012                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACAACACTTTATATATATTTTTATAAATGTAA GGGGTACAAAGGTGCCATTTTGTTACAT60               GGATATACCGTGTAGTGGTGAAGCCTGGGCTTTTAGTGTATCTGTCATCAGAATAACATA120               CGTGTTACCCATAGGAATTTCTCATCACCCGCCCCCTCCACCCTTCGAGTCTCCAATGTC180               CATTCCACAC TCTATATCCACGTGTATGCATATAGCTCCACATATAAGTGAGAACATGTA240              GTATTTGACTTCCTCTTTCTGAGTTATTTCACTTTGATAATGGCCTCCACTTCCATCCAT300               GTTGCTGCAAAAGACATGACCTTATTCTTTTTGATAGCTGGGGAGTACTCCATTG TGTAT360              ATGTACCACATTTCTTTATCCATTCACCCATTGAGAACACTTAGTTGATTCCATATCTTT420               GCTATTGTCACTAGTGCTGCAATAAACATACATGTGCAGGCTCCTTCTAATATACTGATT480               TATATTTTATGGAGAGAGATAGAGTTCTTAGC GAGTGTGCTGTTTATTTCTAGTGTACTT540              GCAACTAATATTCTGTATACTCCCTTTAGGTGATTGGAGATTTAACTTAGATCTCCAGCA600               AGTGCTACAAGAAGAAAAGATCCTGAAGAATCAATCAAGTTTCCGTGAAGTCAAGTCCAA660               GTAACATCCC CGCCTTAACCACAAGCAGGAGAAATGAAGCACATTATCAACTCG714                    MetLysHisIleIleAsnSer                                                         15                                                                            TATGAAAA CATCAACAACACAGCAAGAAATAATTCCGACTGTCCTCGT762                          TyrGluAsnIleAsnAsnThrAlaArgAsnAsnSerAspCysProArg                              101520                                                                        GTGGTTTTGCCGG AGGAGATATTTTTCACAATTTCCATTGTTGGAGTT810                          ValValLeuProGluGluIlePhePheThrIleSerIleValGlyVal                              253035                                                                        TTGGAGAATCTGATCGTCCTG CTGGCTGTGTTCAAGAATAAGAATCTC858                          LeuGluAsnLeuIleValLeuLeuAlaValPheLysAsnLysAsnLeu                              40455055                                                                      CAGGCACCCATGTACTTT TTCATCTGTAGCTTGGCCATATCTGATATG906                          GlnAlaProMetTyrPhePheIleCysSerLeuAlaIleSerAspMet                              606570                                                                        CTGGGCAGCCTATATAA GATCTTGGAAAATATCCTGATCATATTGAGA954                          LeuGlySerLeuTyrLysIleLeuGluAsnIleLeuIleIleLeuArg                              758085                                                                        AACATGGGCTATCTCAAGC CACGTGGCAGTTTTGAAACCACAGCCGAT1002                         AsnMetGlyTyrLeuLysProArgGlySerPheGluThrThrAlaAsp                              9095100                                                                       GACATCATCGACTCCCTGTTTGTC CTCTCCCTGCTTGGCTCCATCTTC1050                         AspIleIleAspSerLeuPheValLeuSerLeuLeuGlySerIlePhe                              105110115                                                                     AGCCTGTCTGTGATTGCTGCGGACCGCTACATC ACCATCTTCCACGCA1098                         SerLeuSerValIleAlaAlaAspArgTyrIleThrIlePheHisAla                              120125130135                                                                  CTGCGGTACCACAGCATCGTGACCATGCG CCGCACTGTGGTGGTGCTT1146                         LeuArgTyrHisSerIleValThrMetArgArgThrValValValLeu                              140145150                                                                     ACGGTCATCTGGACGTTCTGCACGGGGA CTGGCATCACCATGGTGATC1194                         ThrValIleTrpThrPheCysThrGlyThrGlyIleThrMetValIle                              155160165                                                                     TTCTCCCATCATGTGCCCACAGTGATCACC TTCACGTCGCTGTTCCCG1242                         PheSerHisHisValProThrValIleThrPheThrSerLeuPhePro                              170175180                                                                     CTGATGCTGGTCTTCATCCTGTGCCTCTATGTGCAC ATGTTCCTGCTG1290                         LeuMetLeuValPheIleLeuCysLeuTyrValHisMetPheLeuLeu                              185190195                                                                     GCTCGATCCCACACCAGGAAGATCTCCACCCTCCCCAGAGCCAA CATG1338                         AlaArgSerHisThrArgLysIleSerThrLeuProArgAlaAsnMet                              200205210215                                                                  AAAGGGGCCATCACACTGACCATCCTGCTCGGGGTCTTCA TCTTCTGC1386                         LysGlyAlaIleThrLeuThrIleLeuLeuGlyValPheIlePheCys                              220225230                                                                     TGGGCCCCCTTTGTGCTTCATGTCCTCTTGATGACATTC TGCCCAAGT1434                         TrpAlaProPheValLeuHisValLeuLeuMetThrPheCysProSer                              235240245                                                                     AACCCCTACTGCGCCTGCTACATGTCTCTCTTCCAGGTGAAC GGCATG1482                         AsnProTyrCysAlaCysTyrMetSerLeuPheGlnValAsnGlyMet                              250255260                                                                     TTGATCATGTGCAATGCCGTCATTGACCCCTTCATATATGCCTTCCG G1530                         LeuIleMetCysAsnAlaValIleAspProPheIleTyrAlaPheArg                              265270275                                                                     AGCCCAGAGCTCAGGGACGCATTCAAAAAGATGATCTTCTGCAGCAGG1578                           SerProGluLeuArgAspAlaPheLysLysMetIlePheCysSerArg                             280285290295                                                                  TACTGGTAGAATGGCTGATCCCTGGTTTTAGAATCCATGGGAATAACGTTGCCAAG 1634                 TyrTrp                                                                        TGCCAGAATAGTGTAACATTCCAACAAATGCCAGTGCTCCTCACTGGCCTTCCTTCCCTA1694              ATGGATGCAAGGATGACCCACCAGCTAGTGTTTCTGAATACTATGGCCAGGAACAGTCTA1754              TTGTAGGGGCAACTCTATTTGTGACTGGACAG ATAAAACGTGTAGTAAAAGAAGGATAGA1814             ATACAAAGTATTAGGTACAAAAGTAATTAGGTTTGCATTACTTATGACAAATGCATTACT1874              TTTGCACCAATCTAGTAAAACAGCAATAAAAATTCAAGGGCTTTGGGCTAAGGCAAAGAC1934              TTGCTTTCCT GTGGACATTAACAAGCCAGTTCTGAGGCGGCCTTTCCAGGTGGAGGCCAT1994             TGCAGCCAATTTCAGAGT2012                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 297 amino acids                                                   (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLysHisIleIleAsnSerTyrGluAsnIleAsnAsnThrAlaArg                              151015                                                                        AsnAsnSerAspCysPro ArgValValLeuProGluGluIlePhePhe                             202530                                                                        ThrIleSerIleValGlyValLeuGluAsnLeuIleValLeuLeuAla                              3540 45                                                                       ValPheLysAsnLysAsnLeuGlnAlaProMetTyrPhePheIleCys                              505560                                                                        SerLeuAlaIleSerAspMetLeuGlySerLeuTyrLysIleLeuGlu                              6 5707580                                                                     AsnIleLeuIleIleLeuArgAsnMetGlyTyrLeuLysProArgGly                              859095                                                                        SerPheG luThrThrAlaAspAspIleIleAspSerLeuPheValLeu                             100105110                                                                     SerLeuLeuGlySerIlePheSerLeuSerValIleAlaAlaAspArg                              115 120125                                                                    TyrIleThrIlePheHisAlaLeuArgTyrHisSerIleValThrMet                              130135140                                                                     ArgArgThrValValValLeuThrValIleTrpThrPhe CysThrGly                             145150155160                                                                  ThrGlyIleThrMetValIlePheSerHisHisValProThrValIle                              165170 175                                                                    ThrPheThrSerLeuPheProLeuMetLeuValPheIleLeuCysLeu                              180185190                                                                     TyrValHisMetPheLeuLeuAlaArgSerHisThrArgLysIleSer                               195200205                                                                    ThrLeuProArgAlaAsnMetLysGlyAlaIleThrLeuThrIleLeu                              210215220                                                                     LeuGlyValPheIlePheCysTrpAlaP roPheValLeuHisValLeu                             225230235240                                                                  LeuMetThrPheCysProSerAsnProTyrCysAlaCysTyrMetSer                              245250 255                                                                    LeuPheGlnValAsnGlyMetLeuIleMetCysAsnAlaValIleAsp                              260265270                                                                     ProPheIleTyrAlaPheArgSerProGluLeuArgAspAla PheLys                             275280285                                                                     LysMetIlePheCysSerArgTyrTrp                                                   290295                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1108 base pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 133..1026                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..132                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1027..1106                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGG GCCAGAAAGTTCCTGCTTCAGAGCAGAAGATCTTCAGCAAGAACTACAAAGAAGAAA60               AGATTCTGGAGAATCAATCAAGTTTCCTGTCAAGTTCCAGTAACGTTTCTGTCTTAACTG120               CACACAGGAAAGATGAAACACATTCTCAATCTGTATGAAAAC ATCAAC168                          MetLysHisIleLeuAsnLeuTyrGluAsnIleAsn                                          1510                                                                          AGTACAGCAAGAAATAACTCAGACTGTCCTGCTGTGATTTTGCCA GAA216                          SerThrAlaArgAsnAsnSerAspCysProAlaValIleLeuProGlu                              152025                                                                        GAGATATTTTTCACAGTATCCATTGTTGGGGTTTTGGAGAACCTGATG 264                          GluIlePhePheThrValSerIleValGlyValLeuGluAsnLeuMet                              303540                                                                        GTCCTTCTGGCTGTGGCCAAGAATAAGAGTCTTCAGTCGCCCATGTAC312                           Val LeuLeuAlaValAlaLysAsnLysSerLeuGlnSerProMetTyr                             45505560                                                                      TTTTTCATCTGCAGCTTGGCTATTTCCGATATGCTGGGGAGCCTGTAC360                           PhePheIleCysSerLeuAlaIleSerAspMetLeuGlySerLeuTyr                              657075                                                                        AAGATTTTGGAAAACGTTCTGATCATGTTCAAAAACATGGGTTACCTC40 8                          LysIleLeuGluAsnValLeuIleMetPheLysAsnMetGlyTyrLeu                              808590                                                                        GAGCCTCGAGGCAGTTTTGAAAGCACAGCAGATGATGTGGTGGACTCC456                            GluProArgGlySerPheGluSerThrAlaAspAspValValAspSer                             95100105                                                                      CTGTTCATCCTCTCCCTTCTCGGCTCCATCTGCAGCCTGTCTGTGATT504                           LeuPhe IleLeuSerLeuLeuGlySerIleCysSerLeuSerValIle                             110115120                                                                     GCCGCTGACCGCTACATCACAATCTTCCACGCTCTGCAGTACCACCGC552                           AlaAlaAspArgTy rIleThrIlePheHisAlaLeuGlnTyrHisArg                             125130135140                                                                  ATCATGACCCCCGCACCGTGCCCTCGTCATCTGACGGTCCTCTGGGCA600                           IleMetThrP roAlaProCysProArgHisLeuThrValLeuTrpAla                             145150155                                                                     GGCTGCACAGGCAGTGGCATTACCATCGTGACCTTCTCCCATCACGTC648                           GlyCysThr GlySerGlyIleThrIleValThrPheSerHisHisVal                             160165170                                                                     CCCACAGTGATCGCCTTCACAGCGCTGTTCCCGCTGATGCTGGCCTTC696                           ProThrValIle AlaPheThrAlaLeuPheProLeuMetLeuAlaPhe                             175180185                                                                     ATCCTGTGCCTCTACGTGCACATGTTCCTGCTGGCCCGCTCCCACACC744                           IleLeuCysLeuTyrVa lHisMetPheLeuLeuAlaArgSerHisThr                             190195200                                                                     AGGAGGACCCCCTCCCTTCCCAAAGCCAACATGAGAGGGGCCGTCACA792                           ArgArgThrProSerLeuProLysA laAsnMetArgGlyAlaValThr                             205210215220                                                                  CTGACTGTCCTGCTCGGGGTCTTCATTTTCTGTTGGGCACCCTTTGTC840                           LeuThrValLeuLeuGlyVal PheIlePheCysTrpAlaProPheVal                             225230235                                                                     CTTCATGTCCTCTTGATGACATTCTGCCCAGCTGACCCCTACTGTGCC888                           LeuHisValLeuLeuMetThr PheCysProAlaAspProTyrCysAla                             240245250                                                                     TGCTACATGTCCCTCTTCCAGGTGAATGGTGTGTTGATCATGTGTAAT936                           CysTyrMetSerLeuPheGlnVa lAsnGlyValLeuIleMetCysAsn                             255260265                                                                     GCCATCATCGACCCCTTCATATATGCCTTTCGGAGCCCAGAGCTCAGG984                           AlaIleIleAspProPheIleTyrAlaP heArgSerProGluLeuArg                             270275280                                                                     GTCGCATTCAAAAAGATGGTTATCTGCAACTGTTACCAGTAGAATGATT1033                         ValAlaPheLysLysMetValIleCysAsnCysTyr Gln                                      285290295                                                                     GGTCCCTGATTTTAGGAGCCACAGGGATATACTGTCAGGGACAGAGTAGCGTGACAGACC1093              AACAACACTAGGACT1108                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 297 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetLysHisIleLeuAsnLeuTyrGluAsnIleAsnSerThrAlaArg                              15 1015                                                                       AsnAsnSerAspCysProAlaValIleLeuProGluGluIlePhePhe                              202530                                                                        ThrValSerIleValGlyValLeuGluAsn LeuMetValLeuLeuAla                             354045                                                                        ValAlaLysAsnLysSerLeuGlnSerProMetTyrPhePheIleCys                              505560                                                                        SerLe uAlaIleSerAspMetLeuGlySerLeuTyrLysIleLeuGlu                             65707580                                                                      AsnValLeuIleMetPheLysAsnMetGlyTyrLeuGluProArgGly                               859095                                                                       SerPheGluSerThrAlaAspAspValValAspSerLeuPheIleLeu                              100105110                                                                     SerLeuLeuGlySerIleC ysSerLeuSerValIleAlaAlaAspArg                             115120125                                                                     TyrIleThrIlePheHisAlaLeuGlnTyrHisArgIleMetThrPro                              130135 140                                                                    AlaProCysProArgHisLeuThrValLeuTrpAlaGlyCysThrGly                              145150155160                                                                  SerGlyIleThrIleValThrPheSerHisHisValProThrVal Ile                             165170175                                                                     AlaPheThrAlaLeuPheProLeuMetLeuAlaPheIleLeuCysLeu                              180185190                                                                     TyrValHi sMetPheLeuLeuAlaArgSerHisThrArgArgThrPro                             195200205                                                                     SerLeuProLysAlaAsnMetArgGlyAlaValThrLeuThrValLeu                              21021 5220                                                                    LeuGlyValPheIlePheCysTrpAlaProPheValLeuHisValLeu                              225230235240                                                                  LeuMetThrPheCysProAlaAspProTyrCysA laCysTyrMetSer                             245250255                                                                     LeuPheGlnValAsnGlyValLeuIleMetCysAsnAlaIleIleAsp                              2602652 70                                                                    ProPheIleTyrAlaPheArgSerProGluLeuArgValAlaPheLys                              275280285                                                                     LysMetValIleCysAsnCysTyrGln                                                   290295                                                                    

What we claim is:
 1. An isolated DNA molecular having a nucleotidesequence encoding a bovine adrenotropic hormone receptor wherein the DNAsequence is substantially homologous to the sequence in FIGS. 1A-1C (SEQID NO:3).
 2. An isolated DNA molecule having a nucleotide sequenceencoding a human adrenotropic hormone receptor wherein the DNA sequenceis substantially homologous to the sequence in FIGS. 2 (SEQ ID NO:5). 3.An isolated DNA molecule according to claim 1 or 2 wherein the bovine orhuman adrenocorticotropic hormone receptor encoded therein has thebinding properties of a native adrenocorticotropic hormone receptor. 4.A nucleic acid hybridization probe for the detection of mammalianadrenocorticotropic hormone receptor comprising the nucleotide sequenceof claim
 1. 5. A nucleic acid hybridization probe for the detection ofmammalian adrenocorticotropic hormone receptor comprising the nucleotidesequence of claim
 2. 6. A recombinant expression construct comprising anucleotide sequence encoding a bovine or human adrenocorticotropichormone receptor comprising pcDNAI/neo sequences.
 7. A recombinantexpression construct comprising the DNA sequence of claim 1, wherein theconstruct is capable of expressing the bovine adrenocorticotropichormone receptor in a transformed eukaryotic cell culture.
 8. Arecombinant expression construct comprising the DNA sequence of claim 2,wherein the construct is capable of expressing the humanadrenocorticotropic hormone receptor in a transformed eukaryotic cellculture.
 9. A eukaryotic cell culture transformed with the expressionconstruct of claim 7, wherein the transformed eukaryotic cell culture iscapable of expressing bovine adrenocorticotropic hormone receptor.
 10. Aeukaryotic cell culture transformed with the expression construct ofclaim 8, wherein the transformed eukaryotic cell culture is capable ofexpressing the human adrenocorticotropic hormone receptor.